The NMR technique is presently the only technique which permits the observation of the three-dimensional structure of a protein present in a solution and of the movement or activity thereof at a resolution of atomic level. The studies based on the three-dimensional structures of proteins have recently widely been made not only in the fields of the basic research institutions such as those belonging to various universities, but also in the industrial fields including the recent drug-preparation industries. However, the NMR spectrometry is a novel technique developed only about 20 years ago and therefore, it would involve a number of obstructions (problems) to be overcome in the near future. One of such problems is that the NMR spectrometry is limited in the molecular weight of a subject to be analyzed (Non-Patent Document 1).
In the NMR analysis, the increase in the molecular weight of a protein whose structure should be elucidated results in any possible overlapping between NMR signals caused by an increase in the number of signals and any reduction of signal intensities due to the rapid relaxation phenomenon. In particular, if the molecular weight of a target protein exceeds about 20,000, it would be essential to the elimination of any error accompanied by this technique to develop a more precise and advanced NMR technique.
The structural analysis technique which makes use of a highly stable isotope-labeled protein “Stereo-Array Isotope-Labeling technique (SAIL Technique)” is a method wherein any unnecessary structural information obtained in the 1H-NMR analysis is eliminated except for that needed and sufficient for the NMR structural analysis through the exhaustive and selective deuteration of a target protein (SAIL protein) to thus permit the considerable reduction of the time required for the NMR measurement and/or analysis. The methods disclosed in Patent Documents 1 and 2 are techniques which not only allow the highly precise structural elucidation of a target protein, but also permit the automatic structural analysis, with high precision, of even a protein having a molecular weight of about 40,000 higher than the aforementioned upper limit thereof. Thus, there has hereafter been desired for the development of a novel technique which may allow the automatic analysis of fine and precise structure of a high molecular weight protein or a membrane protein, which has a molecular weight of not less than 60 kDa.
In the SAIL technique, the sensitivity of residual signal of a protein is further improved and the limit in the molecular weight of a protein to be analyzed is expanded from the conventional level on the order of 30 kDa to not less than 40 kDa through the efficient increase in the deuteration density of such protein (Non-Patent Document 2). However, the structural analysis of a protein whose molecular weight exceeds 60 kDa cannot easily be analyzed by any simple application of the conventional SAIL amino acids, because of an increase in the signal line width caused by the reduction in the mobility of the protein molecule and of any possible overlapping between NMR signals due to an increase in the number of signals. Moreover, the membrane protein is dissolved in an appropriate medium in the presence of lipids or a detergent, although the molecular weight of the protein per se is not high and therefore, the substantial molecular weight of the membrane protein becomes high and this in turn results in the reduction of the mobility of the protein molecule. Accordingly, the analysis of such a membrane protein through the use of the NMR is not easy like the NMR analysis of the high molecular weight protein. From the aforementioned reasons, there has been desired for the development of any means for obtaining structural information of membrane proteins and high molecular weight proteins, which may presently become subjects for the development of pharmaceutical agents.
Patent Document 1: International Patent Publication WO 03/053910A1;
Patent Document 2: International Patent Publication WO 2005/042469A1;
Non-Patent Document 1: Wuthrich K (1986), NMR of proteins and nucleic acids, Wiley, New York, Wuthrich K (1991); Structural Analysis of Proteins and Nucleic Acid According to the NMR-Two-Dimensional NMR Spectroscopy, Tokyo Kagaku-Dojin Publishing Co., Ltd.;
Non-Patent Document 2: Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Ono M, Guntert P. Nature 2006; in press.